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Alanine Aminotransferase (ALT or
Catalog №
Web: http://htidiagnostics.com HT-A206E-120 HT-A206E-540 SGPT) Reagent Set
HT-A206E-240 HT-A206E-600
High Technology, Inc. HT-A206E-340
INTENDED USE of the control material must be confirmed by the chosen application. Failure to obtain the proper
For the quantitative determination of alanine aminotransferase in serum. range of values in the assay of control material may indicate either reagent deterioration,
instrument malfunction, or procedure errors.
INTRODUCTION
The enzyme alanine aminotransferase is widely reported in a variety of tissue sources. The major CALIBRATION
source of ALT is of hepatic origin and has led to the application of ALT determinations to the study ALT activity is based on the "micromolar extinction coefficient" of NADH at 340 nm (see "Results"
of hepatic diseases. Elevated serum levels are found in hepatitis, cirrhosis, and obstructive section). The instrument manufacturer's calibration guidelines should be followed to calibrate your
jaundice. Levels of ALT are only slightly elevated in patients following a myocardial infarction. analyzer. Assaying the ALT contents of a control serum with known ALT values can be used to
UV methods for ALT determination were first developed by Wroblewski and LaDue in 1956. The assure instrument calibration has been performed correctly.
method was based on the oxidation of NADH by lactate dehydrogenase (LDH). In 1980, the
International Federation of Clinical Chemistry recommended a reference procedure for the RESULTS
measurements of ALT based on the Wroblewski and LaDue procedure. The ALT reagent Values are derived based on the "absorptivity micromolar extinction coefficient" of NADH at 340
conforms to the formulation recommended by the IFCC. nm (0.00622). Units per liter (U/L) of ALT/GPT activity is that amount of enzyme, which oxidizes
one µmol/L of NADH per minute.
PRINCIPLE ΔA/Min Total Volume
The enzymatic reaction sequence employed in the assay of ALT is as follows: U/L = Absorptivity × Sample Volume
ALT ΔA/Min 1.100
L-Alanine + 2-oxoglutarate Pyruvate + L-Glutamate U/L = 0.00622 × 0.100
LDH U/L = ΔA/Min × 1768
+
Pyruvate + NADH + H Lactate + NAD+ + H2O
The pyruvate formed in the first reaction is reduced to lactate in the presence of lactate LIMITATIONS
dehydrogenase and NADH. The activity of ALT is determined by measuring the rate of oxidation of If the ΔA/min. is greater than 0.342, dilute 1 part sample with 9 parts isotonic saline and re-assay.
NADH at 340 nm. Endogenous sample pyruvate is converted to lactate by LDH during the lag Multiply the result by 10. ALT values for neonatal patients have not been established with this
phase prior to measurement. procedure. Grossly icteric or turbid specimen may require the use of a sample blank.
MANUAL PROCEDURE
1. Prepare ALT working reagent according to instructions. Catalog number In vitro diagnostic
2. Pipette 1.0 mL of working reagent into tubes labeled “controls”, patient(s)”, etc.
3. Pre-incubate all tubes at 37°C for at least five minutes.
4. Zero spectrophotometer at 340 nm with distilled water. Temperature Authorized representative
Add 100 µL (0.10 mL) serum to its respective tube, mix gently and turn to a thermos limitation in the European
5. cuvette. Community
6. Read and record absorbance at 1 minute. Continue incubating at 37°C and record Lot number Consult instructions for use
absorbance again at 2 and 3 minutes. Rate should be constant.
7. Determine the average absorbance per minute (ΔA/min), multiply by factor 1768 for results
in U/L. This product conforms to the directive 98/79/EC (IVD-directive)
8. Repeat the procedure for each sample.
NOTE: If cuvette is not temperature controlled, incubate samples at 37°C between readings.
AUTOMATED PROCEDURE
Refer to appropriate application manual available.
QUALITY CONTROL
It is recommended that controls be included in each set of assays. Commercially available control
material with established ALT values may be routinely used for quality control. The assigned value